Homing endonucleases are rare-cutting enzymes that are most often encoded by introns and inteins. They function to cleave DNA and thereby initiate the homing reactions that mobilize their respective genetic elements. Homing enzymes are grouped into four families, based on conserved sequence elements, the LAGLIDADG, GIY-YIG, H-N-H and His-Cys motifs. Inteins are self-splicing proteins that are evolutionarily related to homing endonucleases. Interest in these enzymes is heightened by their phylogenetic diversity and the widespread occurrence of intron and intein homing, coupled with the unusual properties of the endonuclease-nucleic acid interactions. We have made considerable progress in understanding the structure, function and evolution of homing endonucleases and inteins during the past funding period. This work focused on the modularity of these enzymes, on their different modes of catalysis, and on the mechanism of intein action as well as the biotechnological utility of inteins. The proposed work will again combine genetic, biochemical and structural studies to achieve the overall goal of defining the molecular mechanism and evolution of homing endonucleases and inteins. We have selected two endonuclease families, encoded by either group I or group II introns, and an intein for further study. Our endonuclease choices are based on diversity of function of the enzymes, and whether they act independently or in concert with RNA. Our intein work is conducted on the RecA intein from the pathogen Mycobacterium tuberculosis. The four specific aims are as follows: 1. To extend our mechanistic appreciation of the modular GIY-YIG endonucleases, particularly I-TevI; 2. To understand the function of the disparate H-N-H endonuclease family, with I-TevIII and the ribonucleoprotein LtrA as examples; 3. To probe the structural evolution of homing endonucleases, and their acquisition of maturase function; 4. To gain further insight into intein function and to utilize inteins as novel drug targets. Thus, on one hand, our work will continue to expand the understanding of the molecular mechanism and evolution of the functionally and phylogenetically diverse endonucleases that initiate intron homing. On the other hand, the research will shed light on intein function, while exploring the potential of inteins in antimicrobial drug development. [unreadable] [unreadable] [unreadable]